1. Field of the Invention
This invention relates to a method and apparatus for performing novel high-performance liquid chromatography, and more particularly to high-performance liquid chromatography capable of detecting separation patterns in a column.
2. Discussion of the Background
Chromatography is generally classified into gas chromatography and liquid chromatography by the differences in the mobile phase. Liquid chromatography is further categorized into column chromatography (HPLC) and thin layer chromatography (TLC). Recently, high performance thin layer chromatography (HPTLC), which is the same as TLC in principle, has been introduced into the art.
In applications of chromatography as a means of separation, TLC is used mainly for qualitative analysis because of its ease of use, while HPLC is used mainly for quantitative analysis.
The greatest difference between TLC and HPLC is the fact that TLC is an open chromatography system while HPLC is a closed system. In an open system, difficulty is encountered in accurately detecting the sizes and gradation of separated spots due to diffusion of solvents and separated substances. On the other hand, a closed HPLC system, which uses a column of a predetermined volume for separation, is free of the problems mentioned above for TLC so that it is superior in quantitative reproducibility.
Another difference between HPLC and TLC is found in the method of detection after separation. Detection in the open TLC system is via a static method. The spots of the respective components are irradiated with light of a predetermined wavelength after development and separation, either measuring the degree of absorbance of reflected or transmitted light or measuring the fluorescent light intensity after coupling with a fluorescent substance. On the other hand in HPLC, which can be regarded as a dynamic method, the effluent solutes which have been separated in a column according to the retention time of the respective components are detected by measuring the degree of absorbance, fluorescent light intensity or refractive index by means of a detector which is located at the outlet of the column.
Important characteristics in chromatographic analysis are the ability to separate the respective components and the accuracy of the quantitative and qualitative analyses of the separated components. Considering TLC and HPLC from this viewpoint, TLC is disadvantageous since charging of test material in excess of a certain amount is difficult as compared with HPLC and the quantitative analysis is inevitably impaired by the differences in diffusion between the spots of the respective components. On the other hand, TLC has the advantage of an open system in that the entire amount of the charged material can be detected after separation. Namely TLC, in which the charged material is entirely separated and developed on a thin layer sheet, can serve as an excellent means of analysis if accurate quantitative analysis of the respective separated components is available by other means.
On the other hand, in the closed HPLC system, the differences in diffusion experienced in TLC, which depend on the nature of the respective components, seldom occur. Namely, a quantitative analysis of extremely high reproducibility is possible by passing a solution of the components, separately desorbed in the column, through a flow cell of predetermined volume and measuring the light absorbance or fluorescent light intensity of the substances in the flow cell. However, a large problem exists in that sometimes the components of a charged material are not entirely desorbed for separation. Especially in an in-phase HPLC system employing a column packed with silica gel, the residue which remains adsorbed in the column without desorbing cannot be ignored. In order to reduce to a minimum the adsorbed residue in the column, it is the general practice in current HPLC to employ a system which first desorbs out polar substances which would tend to remain adsorbed in the in-phase column, namely, to employ a reverse phase packing material. However, even where a reverse phase column is used, the existence of a residue of a non-polar substance is still probable, making it difficult to desorb out and recover the entire amount of the charge. That is to say, it is difficult to make adequate qualitative and quantitative analyses by conventional HPLC employing the system of detecting and identifying only the substances which are separated and desorbed out of a column. The detection of the adsorbed residual components in a column is impossible regardless of the kind of the packing material. Accordingly, in an analysis of HPLC, one should always consider that a certain percentage of the charged substance remains in the column without being detected. In other words, it is possible with conventional HPLC to make a false determination of subject material present due to failure to detect all of the components of the charged material.
There exists a need, therefore, for a method of high-performance liquid chromatography which does not depend on the detection of eluted subject materials.